SUMMARY At the leading edge membrane of a migrating cell, three membrane-associated master kinases (PKC, PI3K, PDK1) together play a central role in controlling the signaling pathway that regulates chemotaxis up attractant gradients. The present project has provided fundamental insights into key membrane targeting reac- tions, triggered by local Ca2+ and PIP3 signals, that recruit these and other signaling proteins to the leading edge membrane. The resulting membrane-localized signaling network controls cell migration. New studies will focus on the three master kinases and their membrane-based signaling reactions. These studies will elucidate the activation mechanisms of individual kinases and define the pathway connec- tions between them. The lipid bilayer itself is a crucial element of the signaling pathway - the bilayer surface serves as a 2-dimensional scaffold and diffusion platform on which recruited proteins (a) collide with and bind membrane-bound activators and substrates, (b) carry out signaling reactions, and, in some cases (c) combine with other membrane proteins to form higher order signaling complexes. The new Specific Aims investigate the activation mechanisms and regulatory interactions of the three master kinases on planar bilayers. Initial work will analyze each individual membrane-bound kinase to under- stand the sequence of molecular steps leading to kinase activation. Longer term studies will reconstitute all three kinases with their membrane-bound activators and substrates, enabling direct analysis of signal trans- mission and regulatory interactions between membrane-bound components. The project has three Aims: Aim 1. Delineate the molecular steps by which multiple lipids and activators switch on PKC. Aim 2. Elucidate the mechanisms by which lipids, proteins, and oncogenic mutations activate PI3K. Aim 3. Define the interactions between kinases that establish pathway connections and activate PDK1. Completion of the Aims will reveal fundamental molecular principles underlying the activation, signal propagation, and feedback connections of the three master kinases, with major impacts for signaling at the leading edge and diverse areas of signaling biology. Furthermore, each targeted master kinase is directly linked to human cancers and inflammatory disease, thus a mechanistic understanding of kinase activation is medically relevant and could facilitate the development of new therapies.